Experimental infection of an isolate of Leptospira interrogans serovar hardjo in sheep G. Abdollahpour, Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tehran, P O Box 14155-6453, Tehran, Iran. E-mail:
[email protected]
A.W. ENGLISH; J. TASLER, Department of Clinical Sciences, University of Sydney, Private Mail Bag 3, Camden, NSW 2570, Australia.
Abstract Leptospirosis in sheep has been reported from many countries. In contrast to cattle, relatively little experimental work has been carried out in sheep. Sheep and cattle are often graze together, and it is therefore important to investigate the role of sheep in the epidemiology of L. hardjo. Fifteen Merino sheep which were serologically and bacteriologically negative for leptospirosis, were used for this experiment. The sheep were divided into 3 groups of 5 animals each. Group A and B were given 3 conjunctival inoculation of infected urine or culture of Leptospira respectively. Group C were used as control. The results of this experiment showed that group A, which were exposed to the infected urine did not show any serological response. However, there was a significant serological response in all the animals of group B. leptospires were isolated from urine of 3 sheep in group B, whereas all culture isolation attempts failed to isolate leptospires from animals in group B. PCR assay results showed that one sheep in group A and four sheep in group B were shedding L. hardjobovis in their urine. The results of this study confirmed that L. hardjo infection can be easily established in sheep. In this experiment it was also shown that the number of organisms in the inoculum had a great effect on the outcome of the infection. Introduction Leptospirosis in sheep has been reported from many countries. In contrast to the serological prevalence of leptospirosis, outbreaks of clinical leptospirosis in sheep have been infrequently reported. Hartley (1952) associated acute haemolytic disease in Romney ewes and lambs to serovar pomona on the basis of clinical, histological and serological evidence. McCaughan et al., (1980) isolated leptospires from the kidney of a lamb and pooled urine from 6 sheep from two farms where the prevalence of antibodies against serovar hardjo was 76% and 82%, respectively. L. hardjo was also isolated from 3 sheep from another farm that had sporadic losses in 5000 recently introduced wethers (Gordon 1980). Ellis et al., (1984) attributed subacute to chronic multifocal interstitial nephritis with leptospiral infection in Poll Dorset and Southdown rams.
Although cattle are the recognised maintenance hosts for serovar hardjo (Ellis et al., 1981), persistent leptospiruria due to serovar hardjo in sheep where no contact with cattle has occurred, suggests that sheep may be another maintenance host for this serovar (Cousins 1989). Experimental infection of 14 pregnant ewes with serovar hardjo, resulted in mild pyrexia the day after Intra-muscular inoculation and interstitial nephritis or mastitis in 3 ewes (Cousins et al., 1991) Sheep and calves often graze together, therefore, it is important to investigate the role of sheep in the epidemiology of Leptospira interrogans serovar hardjo (herein after referred to as L. hardjo). If sheep are capable of shedding L. hardjo, and transmit the organism to cattle, control programs for L. hardjo will become more complicated. In the present study, an experimental trial was conducted to examine the infectivity and pathogenicity of an isolate of L. hardjo (Abdollahpour et al. 1996) in sheep. This study demonstrats the importance of sheep as a reservoir of L. hardjo infection for cattle. Materials and Methods Fifteen Merino sheep aged 1.5 to 4 years which were serologically and bacteriologically free of leptospira, were used for this study. The sheep were divided into three groups of 5 animals each; Group A. Sheep were given three conjunctival inoculations of urine collected from an infected calf which was shedding L. hardjo, on three consecutive days. Group B. Sheep were give three conjunctival inoculations of 5x108 of L. hardjo culture. Group C. This group were given three conjunctival inoculations, the same way as for group A, but using urine samples collected from a leptospira-free calf. This group was used as control. Sample preparation method: Samples of blood and urine were taken before exposure, at the first day of exposure, then at 3 day intervals for 12 days, and subsequently once a week until the end of experiment. Serological methods: To monitor the antibody titre of animals a standard microscopic agglutination test (MAT), was carried out on all sera as described by Turner (1968). Clinical, Bacteriological and Pathological examinations: Routine clinical examinations were carried out and rectal temperature was recorded once a day, 5 days before exposure until 2 weeks after exposure. Blood samples collected on days 3 to 12 post-exposure were used for culture isolation. Midstream urine samples were cultured prior to the experiment, at the time of exposure, and at weekly intervals throughout the experiment. Urine samples were also examined under a dark-field microscope for presence of leptospires. Animals that showed leptospiruria were euthanised at the end of experiment, portions of the livers and kidneys were cultured for leptospires and also blocks of kidneys were taken for histology and indirect fluorescent antibody (IFA) examinations.
Immunofluorescence examinations: Urine and kiney smears were prepared and examined for presence of leptospira organisms using IFA technique as described by Miller et al. (1989). Polymerase Chain Reaction: The PCR used to detect leptospira genotype hardjobovis DNA in urine samples of sheep was performed as described by Van Eys (1989). Results During the entire experiment observation, non of the sheep in group A, B and C showed clinical sings. No serological response was observed in sheep group A during the entire experiment. However, there was a significant serological response in all the animals of group B. The latest group showed seropositive reaction against serovar hardjo antigen by 14 days after exposure (Table 1). The titres ranged from 1:100 to 1:800 (Figure 1). All sheep in group C remained serologically, bacteriologically and PCR negative during the entire experiment. Table 1: Experimental infection of L. hardjobovis in sheep group A and B: Sheep MAT Titre * No. First Titre Maximum Titre Titre at week 8 PE Culture ---- ---------------------------- ----------477a N N N N 479a N N N N 760a N N N N 779a N N N N 760a N N N N 476b 100 (21) 100 (21) 50 N 483b 200 (12) 800 (21) 400 + (30) 751b 50 (12) 400 (21) 200 + (56) 753b 100 (12) 400 (21) 100 N 757b 100 (14) 200 (30) 200 + (30) ----- ---- -------------- ----------------- ------------------ ----------------- ------------------ --* Titre as reciprocal; a= group A; b= group B; N= negative Days after exposure are shown in parenthesis
Table 2: Incidence of leptospiruria using 3 diagnostic methods in experimentally infected sheep (group A and B): Sheep Age* No. (year) PCR IFA DFE ----- ---------- -----477a 4 N N N 476a 3 N N N 760a 1 32# 32 32, 34, 44 764a 2 N N 44 765a 2 N N 44 476b 3 N N N 483b 1 30, 34 44 44, 56 751b 2 44 24 24, 44 753b 1 34 N N 757b 2 30 N N ----- ---- -------------- ----------------- ------------------ --------------N= negative results; a= group A; b= group B; * approximate # Days after exposures in which each test become positive are shown
Figure 1: Serological reaction of sheep experimentally infected with culture of L. hardjo (group B) 1200
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Leptospires were not isolated from blood or urine from sheep in group A. However, leptospires were isolated from urine of 3 sheep in group B (Table 1) within week 4 and 5 postexposure. Using dark-field examination (DFE), leptospiral organisms were found in urine samples of one animal in group A and two animals in group B during weeks 4 to 5 post exposure. However, using the IFA method, leptospira organisms were demonstrated in urine samples of only 2 sheep of group B in week 4 post exposure (table 2) and none of the animals in group A. Four of the animals were euthanised for histopathological examinations. No macroscopic changes related to leptospirosis was found in the animals. PCR assay results showed that 4 animals in group B and one animal were shedding L. hardjobovis in their urine during week 4 to 5 after exposure (table 2).
Discussion The results of this experiment show that L. hardjo infection can be established in sheep either by exposing the animals to the urine of an infected calf or to pure culture of L. hardjo. However, those animals which were exposed to the culture of the organism produced a much higher level of leptospiruria and also a detectable serological response. This was perhaps because of larger number of organisms present in the culture which stimulated a much higher
immune response than occurred using infected urine which contained lower numbers of the organism. However, serological responses to inoculation depend on other factors as well, including the virulence of the infecting organism, the immune status and the age of the host. The results of the present experiment also showed that younger sheep are more susceptible to infection than older sheep. Although Cousins et al. (1991) reported that the most effective route for experimental infection of sheep was IM injection of the organisms, in the present experiment, the conjunctival route was used, because this has been reported to be the most likely route of natural infection (Thiermann and Handsaker, 1985). In the present study 7 out of 10 sheep exposed to the organism were shedding leptospires in the urine in a period of from 1 to 32 days. The culture results also showed that the L. hardjo strain used for this experiment colonised the kidneys and established leptospiruria in 3 sheep of group B. It is therefore extremely possible that these sheep would be able to transmit the infection to cattle populations under field conditions. However, despite the demonstration of L. hardjo infection in these sheep, none of them showed any clinical symptoms during the course of experiment. The biphasic nature of MAT titres in 3 sheep is similar to that observed in the cows. Although there was a significant correlation between the culture results and the biphasic pattern, further studies are required to determine the significant of this correlation under field condition. In this experiment the highest MAT titre was 1:800, whereas, in overseas studies experimental infection of sheep with L. hardjo (Andreani et al., 1983) resulted in a much higher serological titre (1:500000). This difference is possibly because of the different experimental conditions or low pathogenicity of L. hardjo. A comparison between group A and B provides some interesting information about the influence of the type of inoculum on the subsequent responses of the animals. Sheep exposed to the urine of an infected calf did not develop any serological response, and despite demonstration of the organism in the urine of 3 sheep ( tabl2 2), leptospires were not isolated from the urine of this group . On the other hand, all animals in group B which were exposed to the culture of L. hardjo , developed serological titres against L. hardjo, and had a more prolonged and higher concentration of leptospiruria than those in group A. Leptospires were isolated only from urine of 3 sheep of group B, perhaps the high number of organisms in the inoculum for group B provoked a serological response and higher concentration of leptospiruria. Leptospiruria in group B was much higher and leptospires were seen on several occasions during the experiment. References: Abdollahpour G; English A W and Taslar I: Isolation of Leptospira interrogans serovar grippotyphosa from a heifer in New South Wales. Australian Veterinary Journal 1996(73) 3:109-110
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